Fervidobacterium amylase and pullulanase

ABSTRACT

The present invention relates to Fervidobacterium amylase and pullulanase preparations and their use in producing sweeteners and ethanol from starch. In particular the enzymes are derived from Fervidobacterium pennavorans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK95/00095 filed Mar. 2, 1995, which is incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to a novel thermostable amylase and anovel thermostable pullulanase and their use in the production ofsweeteners and ethanol from starch.

BACKGROUND OF THE INVENTION

The production of sweeteners from starch has been largely improved byapplication of different microbial enzymes to obtain better quality andyields, but the necessity of performing several steps of thestarch-hydrolysing process at elevated temperatures means that there isstill a need for new starch-hydrolysing enzymes with increased thermalstability.

It is known that Pyrococcus, e.g. Pyrococcus wosei and Pyrococcusfuriosus, for reference see Arch. Microbiol. 155, 1991, pp. 572-578, andAppl. Env. Microbiol. 56, 1990, pp.1985-1991, can produce highlythermostable amylases.

It is the object of this invention to provide an amylase and apullulanase with temperature optimum at 80° C. or above 80° C.

SUMMARY OF THE INVENTION

We have unexpectedly found that a novel thermostable amylase can beobtained from the genus Fervidobacterium, a genus not previouslyreported to produce thermostable amylase; and a novel thermostablepullulanase which can be obtained from Fervidobacterium pennavorans;these new enzymes have temperature optimum around 80°-90° C.

Accordingly, the invention provides an amylase preparation,characterized by being producible by cultivation of an amylase producingstrain of the genus Fervidobacterium, and a pullulanase preparation,characterized by being producible by cultivation of a pullulanaseproducing strain of Fervidobacterium pennavorans.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 shows the relative activity (% rel.) of an amylase (¤) and apullulanase (▪) of the invention at various temperatures (determined atpH 5.5 with starch and pullulan, respectively, as substrate).

FIG. 2 shows the relative activity (% rel.) of an amylase (¤) and apullulanase (▪) of the invention at various pH, determined at 90° C.with starch and pullulan, respectively, as substrate.

DETAILED DISCLOSURE OF THE INVENTION The Microorganism

According to the invention, amylase is derived from an amylase producingstrain of the genus Fervidobacterium, in particular Fervidobacteriumpennavorans, and pullulanase is derived from a pullulanase producingstrain of Fervidobacterium pennavorans.

A strain representative of Fervidobacterium pennavorans has beendeposited according to the Budapest Treaty on the InternationalRecognition of the Deposits of Microorganisms for the Purpose of PatentProcedures, on 12 Mar., 1992, at Deutsche Sammlung von Microorganismenund Zellkulturen GmbH, under Accession No. DSM 7003.

Production of Amylase and Pullulanase

Amylase and pullulanase of the invention may be produced by anaerobiccultivation of the above mentioned strain on a nutrient mediumcontaining suitable carbon and nitrogen sources, such media being knownin the art. Anaerobic conditions may be achieved during the preparationof media by sparging with N₂ and following the anaerobic techniques asdescribed by Balch and Wolfe in Appl. Env. Microbiol. 32, 1976, pp.781-791.

Alternatively, amylase and pullulanase of the invention can be producedby aerobic cultivation of a transformed host organism containing theappropriate genetic information from the above mentioned strain. Suchtransformants can be prepared and cultivated by methods known in theart.

The amylase and the pullulanase may be recovered by removing the cellsfrom the fermentation medium (e.g. by centrifugation or filtration) andthen concentrating the broth (e.g. by ultrafiltration). If desired, theamylase and the pullulanase may be further purified by known methods.

Immunochemical Properties

The enzymes of the invention have immunochemical properties identical orpartially identical (i.e. at least partially identical) to those of anenzyme derived from the strain Fervidobacterium pennavorans, DSM 7003.

The immunochemical properties can be determined immunologically bycross-reaction identity tests. The identity tests can be performed bythe well-known Ouchterlony double immunodiffusion procedure or by tandemcrossed immunoelectrophoresis according to Axelsen N. H.; Handbook ofImmunoprecipitation-in-Gel Techniques; Blackwell Scientific Publications(1983), chapters 5 and 14. The terms "antigenic identity" and "partialantigenic identity" are described in the same book, Chapters 5, 19 and20.

Monospecific antisera are generated according to the above mentionedmethod by immunizing rabbits with the purified enzymes of the invention.The immunogens are mixed with Freund's adjuvant and injectedsubcutaneously into rabbits every second week. Antisera are obtainedafter a total immunization period of 8 weeks, and immunoglobulins areprepared therefrom as described by Axelsen N. H., supra.

The Enzymes

An amylase of the invention can be characterized by having amylaseactivity at temperatures of from below 60° C. to approximately 120° C.,having activity optimum at temperatures in the range 85°-95° C.,determined at pH 5.5 with starch as substrate. The amylase can also becharacterized by having amylase activity at pH values of from below pH4.5 to approximately pH 9.8, having optimum in the range pH 5.0 to pH6.0, determined at 90° C. with starch as substrate.

A pullulanase of the invention can be characterized by havingpullulanase activity at temperatures of from below 60° C. toapproximately 100° C., having activity optimum at temperatures in therange 75°-85° C., determined at pH 5.5 with pullulan as substrate. Thepullulanase can also be characterized by having pullulanase activity atpH values of from below pH 4.5 to approximately pH 9.8, having optimumin the range pH 5.0 to pH 6.0, determined at 90° C. with pullulan assubstrate.

Determination of Amylase Activity

Amylase activity is determined by measuring the amount of reducing sugarreleased during the incubation with starch. One unit (U) of amylaseactivity is defined as the amount of amylase that releases 1μ mole ofreducing sugar (as maltose standard) per min. under the following assayconditions: A 0.05 ml volume of 1% soluble starch is added to 0.05 ml of0.1M sodium acetate buffer pH 5.5. 25 μl of enzyme solution are added tothis mixture and the sample is incubated at 90° C. for 30 min. Thereaction is stopped by cooling on ice, and the amount of reducing sugaris determined by dinitrosalicylic acid. Sample blanks are used tocorrect for nonenzymatic release of reducing sugar.

Determination of Pullulanase Activity

Pullulanase activity is determined by measuring the amount of reducingsugar released during the incubation with pullulan. One unit (U) ofpullulanase activity is defined as the amount of pullulanase thatreleases 1μ mole of reducing sugar (as maltose standard) per min. underthe following assay conditions: A 0.05 ml volume of 1% pullulan is addedto 0.05 ml of 0.1M sodium acetate buffer pH 5.5. 25 μl of enzymesolution are added to this mixture and the sample is incubated at 90° C.for 30 min. The reaction is stopped by cooling on ice, and the amount ofreducing sugar is determined by dinitrosalicylic acid. Sample blanks areused to correct for nonenzymatic release of reducing sugar.

Industrial Applications

The enzymes of this invention possess valuable properties allowing forvarious industrial applications. In particular the enzymes, in beingthermostable, find potential application in the production of sweetenersand ethanol from starch. Conditions for conventional starch convertingprocesses and liquefaction and/or saccharification processes aredescribed in for instance U.S. Pat. No. 3,912,590 and EP patentpublications Nos. 252,730 and 63,909.

The following example further illustrates the present invention, and itis not intended to be in any way limiting to the scope of the inventionas claimed.

EXAMPLE 1 Cultivation

The strain Fervidobacterium pennavorans, DSM 7003, was recultured fromglycerol-preserved cells using the medium recommended by the DeutscheSammlung von Mikroorganismen (DSM). The microorganisms were grown in 1liter batch cultures under the following conditions:

Medium

NH₄ Cl: 0.5 g/l

MgSO₄,7H₂ O: 0.16 g/l

K₂ HPO₄,H₂ O: 1.6 g/l

NaH₂ PO₄,H₂ O: 1.0 g/l

yeast extract: 1.0 g/l

trypticase: 2.0 g/l

Before autoclaving 10 ml/l of vitamin solution, 10 ml/l of Trace elementsolution, 0.05% cystein, HCl and 0.0002% of resazurine were added.Before inoculation, sterile Na₂ S,9H₂ O was added at a concentration of0.05%.

In the medium sulphur and tryptone were omitted and starch (0.5% w/v)was added as the only carbohydrate. Cultivation was performed at pH 6.5and at 70° C. The cell density achieved in this medium was ≧10⁸cells/ml. Anaerobic conditions were achieved during the preparation ofmedia by sparging with N₂ and following the techniques as described byBalch in Appl. Env, Microbiol. 32, 1976, pp. 781-791.

After cultivation the culture fluid was centrifuged at 12.000×g for 30min. at 4° C., and the cell free supernatant was concentrated up to100-fold using an Amicon Ultrafiltration System. The cell pellet wasresuspended in 50 mM sodium acetate buffer pH 5.5 and sonicated threetimes for 3 min. at 50% duty cycle by a BRANSON 450 sonifier. The celldebris was separated from the supernatant after centrifugation at10.000×g for 30 min. at 4° C.

The following total activity (U) in both supernatant and cell extractwas found:

Amylase activity: 2.5 U/l

Pullulanase activity: 4.5 U/l

The half-life, determined as the incubation time (min) at 85° C. afterwhich 50% of the original activity is detected, gave the followingresults:

Amylase activity: 40 min

Pullulanase activity: 40 min

Temperature Optima

Temperature optima were determined by incubation of samples for 30minutes at pH 5.5 at temperatures from 60° C. to 120° C. The incubationwas conducted in closed Hungate tubes in order to prevent boiling of thesolution.

FIG. 1 shows the result (Amylase (¤) and pullulanase (▪)).

pH Optima

To determine pH optima, Universal buffer (Britten and Robinson) was usedto obtain values from pH 4.0 to pH 10.0. Samples were incubated for 30minutes at 80° C. at the pH in question.

FIG. 2 shows the result (Amylase (¤) and pullulanase (▪)).

We claim:
 1. An isolated amylase obtained from an amylase producingstrain of the genus Fervidobacterium, having (a) a pH optimum in therange of 5.0 to pH 6.0, determined at 90° C. with starch as substrate,and (b) a temperature optimum in the range of 85° to 95° C., determinedat pH 5.5 with starch as substrate.
 2. The isolated amylase of claim 1,wherein the amylase producing strain is a Fervidobacterium pennavoransstrain.
 3. The isolated amylase of claim 2, wherein the amylaseproducing strain is Fervidobacterium pennavorans, DSM
 7003. 4. A methodfor producing sweeteners, comprising reacting starch with an isolatedamylase according to claim 1 wherein a sweetener is generated, andisolating the sweetener.
 5. A method for producing ethanol, comprisingreacting starch with an isolated amylase according to claim 1 whereinethanol is generated, and isolating the ethanol.